H5N1 cold-adapted (ca) vaccines: Three candidate vaccines were developed based on H5N1 viruses isolated in 1997, 2003 and 2004. In each virus, the HA and NA genes were derived from wild-type H5N1 viruses, and the six internal protein gene segments came from the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. The HA in each case was modified to remove the multibasic amino acid cleavage site that is a virulence motif. We performed open-label trials to evaluate the safety, infectivity, and immunogenicity of H5N1 VN 2004 AA ca and H5N1 HK 2003 AA ca. The H5N1 VN 2004 AA ca vaccine virus was evaluated at dosages of 106.7 TCID50 and 107.5 TCID50, and the H5N1 HK 2003 AA ca vaccine was evaluated at a dosage of 107.5 TCID50. Two doses were administered intranasally to healthy adults in isolation at 4 to 8 week intervals. Vaccine safety was assessed through daily examinations, and infectivity was assessed by viral culture and by realtime reverse transcription-polymerase chain reaction testing of nasal wash (NW) specimens. Immunogenicity was assessed by measuring hemagglutination-inhibiting (HI) antibodies, neutralizing antibodies, and IgG or IgA antibodies to recombinant (r)H5 VN 2004 hemagglutinin (HA) in serum or NW. Fifty-nine participants were enrolled: 21 received 106.7 TCID50 and 21 received 107.5 TCID50 of H5N1 VN 2004 AA ca and 17 received H5N1 HK 2003 AA ca. Shedding of vaccine virus was minimal, as were HI and neutralizing antibody responses. Fifty-two percent of recipients of 107.5 TCID50 of H5N1 VN 2004 AA ca developed a serum IgA response to rH5 VN 2004 HA. The live attenuated H5N1 VN 2004 and HK 2003 AA ca vaccines bearing avian H5 HA antigens were very restricted in replication and were more attenuated than seasonal LAIV bearing human H1, H3 or B HA antigens. The H5N1 AA ca LAIV elicited serum ELISA antibody but not HI or neutralizing antibody responses in healthy adults. H7N3 cold-adapted (ca) vaccine: Based on promising preclinical data in mice and ferrets, a Phase I study was undertaken to evaluate the safety, level of replication, infectivity and immunogenicity of an H7N3 ca vaccine. The H7N3 BC 2004/AA ca virus is a live attenuated, cold-adapted, temperature-sensitive influenza virus derived by reverse genetics from the wild-type low pathogenicity avian influenza virus A/chicken/British Columbia/CN-6/2004 (H7N3) and the A/AA/6/60 ca (H2N2) virus. We evaluated the safety, infectivity, and immunogenicity of two doses of 107.5 TCID50 of the vaccine administered by nasal spray 5 weeks apart to normal healthy seronegative adult volunteers in an inpatient isolation unit. The subjects were followed for 2 months after 1 dose of vaccine or for 4 weeks after the second dose. Twenty-one subjects received the first dose of the vaccine, and 17 subjects received two doses. The vaccine was generally well tolerated. No serious adverse events occurred during the trial. The vaccine was highly restricted in replication: 6 (29%) subjects had virus recoverable by culture or by rRT-PCR after the first dose. Replication of vaccine virus was not detected following the second dose. Despite the restricted replication of the vaccine, 90% of the subjects developed an antibody response as measured by any assay: 62% by HI assay, 48% by microneutralization assay, 48% by ELISA for H7 HA-specific serum IgG or 71% by ELISA for H7 HA-specific serum IgA, after either one or two doses. Following the first dose, vaccine-specific IgG secreting cells as measured by ELISPOT increased from a mean of 0.1 to 41.6/1,000,000 PBMCs;vaccine specific IgA secreting cells increased from 2 to 16.4/1,000,000 PBMCs. The antibody secreting cell response after the second dose was less vigorous, which is consistent with the observed low replication of vaccine virus after the second dose and consequent lower antigenic stimulation. The live attenuated H7N3 vaccine was well tolerated but was highly restricted in replication in healthy seronegative adults. Despite the restricted replication, the vaccine was immunogenic, with serum IgA being the most sensitive measure of immunogenicity. A study to evaluate the safety, level of replication, infectivity and immunogenicity of a single dose of the H7N3 ca vaccine was planned. However, the study was not initiated as planned because the 2009 pandemic H1N1 virus appeared and has continued to circulate in the community. H2N2 cold-adapted (ca) vaccine: H2N2 viruses caused the 1957 influenza pandemic and circulated in humans until 1968 when they were replaced by H3N2 viruses. Although H2 viruses have not circulated in humans since 1968, this subtype is maintained in avian reservoirs worldwide. An H2 vaccine is a high priority in preparing for a pandemic because H2 viruses have a proven capability for causing disease in humans and persons born after 1968 lack H2-specific immunity. Because the currently licensed live attenuated influenza vaccine utilized in the United States bears the internal protein genes of the influenza A/AnnArbor/6/60 (H2N2) ca virus, the A/AnnArbor/6/60 (H2N2) ca virus was a logical first choice for evaluation as an H2 vaccine candidate. However, this virus with the H2 HA and N2 NA has not been evaluated in seronegative people. An IND was submitted for a Phase I study to evaluate safety, level of replication, infectivity and immunogenicity of A/Ann Arbor/6/60 ca (H2N2) virus. Twenty-one subjects received the first dose and 18 received two doses of the vaccine. The vaccine was well-tolerated and the analysis of the data is in progress. H6N1 cold-adapted (ca) vaccine: Based on promising preclinical data in mice and ferrets, an IND was submitted for a Phase I study to evaluate safety, level of replication, infectivity and immunogenicity of an H6N1 ca vaccine based on A/teal/Hong Kong/W312/1997 (H6N1). Twenty-two subjects received the first dose and 18 received two doses of the vaccine. The vaccine was well-tolerated and the analysis of the data is in progress.